Research Paper Volume 12, Issue 16 pp 16083—16098

Figure 4. Inhibition of autophagy recapitulates the effect of chidamide. (A) Immunoblotting analysis of poly (ADP-ribose) polymerase (PARP) in primary CLL cells after chloroquine (CQ, 10μmol/L) treatment for 24 hours. Shown are 2 representative blots from the samples of 6 patients. The bar graph represents the relative PARP cleavage/GAPDH ratio measured by immunoblotting. (B, C) Flow cytometry using Annexin V–FITC/PI staining for cell apoptosis analysis. Primary CLL cells were incubated with 10μmol/L chloroquine for 24 hours while MEC-1 cell line were incubated with indicated concentrations of chidamide for 24 hours. Representative data shown are from 9 patients and three independent experiments of MEC-1. The bar graphs showed the percentage of apoptotic cells. (D) CCK8 assay for detecting metabolically active cells. MEC-1 cell lines was incubated with indicated concentrations of chloroquine for 24 and 48 hours. Viability of cells compared with the corresponding controls was shown from three independent experiments. (E, F) MEC-1 cells were transfected with ATG5 or nontargeting scrambled siRNA as indicated. Expression levels of targeted gene were analyzed by Immunoblotting. Percentage of apoptotic cells was assessed by flow cytometry following Annexin V-FITC/PI staining.